Biochip device

ABSTRACT

A biochip device includes a stratified structure, a light coupler, and an optofluidic portion. The stratified structure includes a top layer having a refractive index n 1 , a bottom layer having a refractive index n 3 , and an intermediate layer between the top and bottom layers having a refractive index n 2 . The light coupler optically couples a light source and the top layer to generate waves that are guided in a plurality of directions inside the top layer. The optofluidic portion is supported on a surface of the top layer and includes a hybridizing chamber containing a hybridizing solution and pads supported on the surface of the top layer and situated within the hybridizing chamber. Probe molecules are deposited on the pads. The refractive index n 2  of the intermediate layer is greater than or equal to a highest index of refraction of the hybridizing chamber and the hybridizing solution.

CROSS REFERENCE OF RELATED APPLICATION

This application a continuation claiming benefit under 35 U.S.C. § 120of the filing date of U.S. patent application Ser. No. 16/205,256 filedNov. 30, 2018, which is a continuation claiming benefit under 35 U.S.C.§ 120 of the filing date of U.S. patent application Ser. No. 13/976,596filed Oct. 7, 2013, now U.S. Pat. No. 10,184,891, which is a 371 ofPCT/FR2011/053208, filed Dec. 28, 2011, which claims priority to FrenchApplication No. 1061349, filed Dec. 29, 2010, the respectivedisclosure(s) of which is(are) incorporated herein by reference.

FIELD OF THE DISCLOSURE

The invention relates to a biochip device for analyzing biologicalmolecules by fluorescent marking.

BACKGROUND

In biochip devices, a substrate includes pads constituted by probemolecules capable of hybridizing in preferential manner with targetmolecules contained in a hybridizing solution obtained from a sample tobe analyzed. The target molecules are marked with the help ofchromophore elements capable of emitting fluorescence when they areexcited by appropriate light, the wavelength of the fluorescencedepending on the nature of the chromophore elements.

After hybridizing, the biochip is dried and illuminated with a lightsource at the excitation wavelength of the chromophores marking thetarget molecules, and an image of the fluorescence of the biochip ispicked up with the help of appropriate objects. In the image obtained inthis way, the intensity of each point is associated with the quantity ofchromophores present at the corresponding point of the biochip and thusassociated with the number of target molecules that have beenselectively fixed at that point during the hybridizing stage thus makingit possible to obtain information about the biological species contentof the hybridized solution.

That type of sequential reading of the fluorescence of the biochip afterhybridizing is nevertheless unsuitable for performing real time readingof the hybridizing signal since the stages of hybridizing and of imagetaking are spaced apart in time, and take place in separate hybridizingand reading appliances.

Certain appliances are capable of performing both the hybridizing andthe reading stages, thus making it possible to detect the signal in realtime during the hybridizing stage (see in particular Y. Marcy, P. Y.Cousin, M. Rattier, G. Cerovic, G. Escalier, G. Bena, M. Gueron, L.McDonagh, F. L. Boulaire, H. Benisty, C. Weisbuch, J. C. Avarre,“Innovative integrated system for real time measurement of hybridizationand melting on standard format microarrays” Biotechniques 44, 2008,913). The image of the fluorescence of the pads carrying the hybridizedmolecules is acquired in the presence of the hybridizing liquidcontaining the target molecules that are marked, and thus fluorescent,and they may be present at high concentration. Fluorescence is thenobserved coming simultaneously from the target molecules attached to thepads of probe molecules (forming the useful signal) and from fluorescentmolecules in the solution (constituting a background signal that isadded to the useful signal).

That is disadvantageous, since the strong background signal generated bythe fluorescent species in solution limits the sensitivity with which itis possible to detect the attachment of target molecules and limits thedynamic range over which hybridization can be measured.

In order to avoid that drawback, one possibility consists in selectivelyexciting the molecules at the surface of the biochip without excitingthe molecules present in the solution, by using an evanescent wave atthe surface of the biochip so as to excite only the fluorescent pads(one technique often used for that purpose is a configuration of thetotal internal reflection fluorescence (TIRF) type). By way of example,other evanescent wave excitation methods consist in using substratescarrying a waveguide, preferably a monomode waveguide, and in excitingone or more modes in the waveguide with the help of etched couplinggratings or in exciting guided modes in the biochip by lighting via anedge face (US 2004/077099 A1).

In general, it is also necessary to take into consideration theinteraction between the guided waves and the optofluidic portion of thedevice in contact with the waveguide.

For the above-described evanescent wave devices, light coupling makes itnecessary to use excitation devices having mechanical constraints thatare very demanding in terms of precision.

That type of coupling makes it necessary either to use optical systemswith sub-micrometer precision on polished edge faces for coupling theexcitation light to a single mode, or else to have recourse to beamsthat are collimated with very precise angles (a few milliradians orless).

Nevertheless, it is known that incident light on non-uniform bodies suchas metallic or dielectric particles, or more generally diffusers, makeit possible to excite guided modes of any planar structure providing theelements of the diffuser are positioned very close to the waveguide, inthe evanescent tail of the modes. This makes it possible to avoid thetight coupling tolerances encountered with the above-described devices.Such diffusers are referred to herein as “substantially non-directionalmeans for generating or coupling guided modes”.

The term “substantially non-directional coupling means” is used hereinto designate means for coupling excitation light into the waveguide inthe form of waves that are guided in a plurality of directions insidethe waveguide by using excitation light coming from a plurality ofdirections. The excitation light may be coupled with the waveguide byusing an excitation light beam that is not necessarily collimated. Withsuch coupling means, there is no longer any need for the beam to beoriented very precisely relative to the waveguide.

Such coupling means are known for waveguides and solar cells, e.g., madeof silicon. In those applications, a diffusing disordered interfaceserves to transform the incident light into guided light so that it isused in the waveguide or absorbed in the solar cell. For waveguideapplications, the purpose is then to use the light in the waveguide sothat it is absorbed therein, e.g., for use in a photodetector device.For solar cells, diffusion takes place over the entire surface of thecell in order to be able to capture all of the light intercepted by thecell.

SUMMARY

An object of the invention is to provide a simple solution to theabove-mentioned problems of biochip devices known in the prior art.

To this end, the invention provides a biochip device comprising asubstrate constituted by at least one plate of material forming amultimode waveguide and carrying chromophore elements suitable foremitting fluorescence in response to excitation by guided waves havingan evanescent portion, the device being characterized in that itincludes coupling means for coupling excitation light with the waveguidein the form of guided waves, the coupling means being substantiallynon-directional.

Integrating substantially non-directional coupling means in a biochipdevice makes it possible to avoid the precision constraints encounteredin the prior art.

In the invention, the coupling means cover only a portion of thebiochip. In particular, the coupling means are placed at a distance fromthe fluidic or optofluidic portion so as to avoid extracting guidedwaves into the fluid containing fluorescent molecules, which isprecisely what it is sought to avoid by exciting fluorophores that areexcited by the evanescent waves only.

In an advantageous configuration, the device includes mode filter meansfor eliminating from the waveguide guided modes having an effectiveindex less than or equal to a predetermined threshold value, thisthreshold value being selected so that no guided mode escapes from thewaveguide beyond the zone having the mode filter means.

A first drawback of approaches based on substantially non-directionalcoupling means lies in the low efficiency of the coupling of theexciting modes with the guided modes. In order to reach a given guidedmode intensity, it is possible to use an exciting source that is moreintense. Nevertheless, the main drawback with a multimode waveguide isthat that type of method of exciting guided modes tends to excite modesregardless of their effective index. Unfortunately, modes with smallereffective indices correspond to modes that leave the waveguide andpenetrate into the fluid or into the optofluidic portions, where theycontribute to increasing the background signal.

Because the guided modes transfer a propagating flux into the fluid onlyon contact with the fluid or the optofluidic portion, the use ofnon-directional coupling means can advantageously be combined with modefilter means that eliminate the unwanted modes that are capable ofinteracting with the fluid or the optofluidic portion.

From a theoretical point of view, the condition for non-transfer of aguided mode to an interface is conventionally presented in the form ofan angle (angle of incidence at the interface being greater than acritical angle), however in more fundamental terms this condition can beexpressed in the form of an effective index of the guided mode, whicheffective index must be greater than that of the fluid or of theoptofluidic portion.

Generalizing from the above propositions, in the device of theinvention, easy mechanical coupling is provided by means for generatingguided waves that are low directional, such as optical diffusing mediain particular, whereas the mode filter means serve to selectively filterout those of the guided modes that can be extracted from the waveguideand thereby increase the interfering background signal. Thus, the guidedmodes of effective index that is less than that of the material of thehybridizing chamber and less than that of the biological solution arefiltered out before they reach the optofluidic zone and a fortioribefore they reach the zone carrying the chromophore elements, therebyavoiding exciting free chromophore elements in solution and out of reachof the evanescent wave.

In a first embodiment, the mode filter means comprise an extraction, orfilter, layer in contact with the waveguide and formed by a medium ofindex substantially equal to the predetermined threshold value so as tofilter the guided modes of effective index less than the threshold valueby extracting them, so that they do not reach the zones includingfluidic or optofluidic functions. The extraction layer determines theabove-mentioned threshold value below which all previously-guided modesare extracted from the waveguide.

In a variant, the extraction layer is interposed between the waveguideplate and an absorption bottom layer, the extraction layer and theabsorption layer extending substantially along the entire length of thewaveguide, the absorption layer having an index not less than that ofthe extraction intermediate layer and presenting absorption at theexcitation wavelength of the chromophores that is considerable at thescale of the light path between the coupling means and a zone of thewaveguide carrying the chromophore elements.

In practice, the absorption layer has an absorption coefficient that isgreater than or equal to 2/L, where L corresponds to the distancebetween the non-directional coupling means and the optofluidic portion,in order to guarantee sufficient absorption of the modes that are ofindex less than the predetermined threshold value. These modes arecaused to propagate in this layer and after a path length of about Lthey present transmission of less than exp(−2), which is approximatelyequal to 0.14. The waveguide plate and the extraction layer make itpossible with thicknesses known to the person skilled in the art to haveat least one guided mode over at least one length L for which the indexneff is greater than the desired threshold.

In another variant of this first embodiment, the mode filter means arecarried by the waveguide and are located between a zone of the waveguidein which the guided waves are generated and a zone of the waveguidecarrying the chromophore elements.

Advantageously, absorption means or deflector means for absorbing ordeflecting the guided modes extracted from the waveguide are placed onthe extraction layer, so that the extracted modes cannot reach thefluidic or optofluidic portions of the biochip.

By way of example, the absorption means consists in a wideband filter.The deflector means may consist in a prism or in a grating, these meansbeing directly in contact with the extraction layer.

According to another characteristic of the invention, the extractionlayer and the absorption means or the deflector means extend along thewaveguide over a distance that is longer than the length that makes itpossible for the guided mode for filtering that has the greatesteffective index to interact at least once with the interface throughwhich the modes are filtered. This distance is given by 2×e×tan Θ, wheree is the thickness of the waveguide and Θ is the reflection angle insidethe waveguide and relative to the normal of the waveguide. With such aminimum extent for the mode filter means, it is guaranteed that all ofthe guided modes of effective index less than the predeterminedthreshold value are subjected at least to refraction or to absorption orto deflection at the interface with the waveguide and are thus extractedfrom the waveguide.

According to another characteristic of the invention, the mode filtermeans extend upstream from and outside the zone carrying the chromophoreelements and also in part in said zone. This has the advantage offiltering photons that might have been diffused by the edges of thehybridizing chamber to produce guided modes in the waveguide ofuncontrolled index that might subsequently leave the waveguide andexcite the hybridizing solution.

Preferably, the threshold value is selected to be greater than or equalto the greatest refractive index of the elements constituting theenvironment of the chromophores and that are generally in opticalcontact, such as, for example, the elements constituting a hybridizingchamber placed on the substrate and a hybridizing fluid contained in thechamber, thereby avoiding any guided modes of effective index less thanthe threshold value being extracted from the waveguide and propagatingdirectly into the hybridizing fluid, or else indirectly into the fluidvia the material of the hybridizing chamber, where they would excite thechromophores of target molecules that are not attached to probemolecules.

In practice, the threshold value lies in the range n=1.30 to n=1.45since the refractive index of a hybridizing solution generally lies inthe range n=1.3 to n=1.4 and the material constituting the chamber isusually polydimethylsiloxane (PDMS) for which n=1.42.

In a second embodiment, the mode filter means are formed by the platecarrying the chromophore elements and having top and bottom faces thatdiverge from each other going from the zone of the coupling means todownstream from the zone carrying the chromophore elements, so as toraise the smallest effective index in the light being distributed on theoccasion of each internal reflection. This thus corresponds to makingthe rays of the guided waves more oblique on reaching the optofluidiczone than the limit angle associated with passing into the fluid or intothe material of the hybridizing chamber.

In this second embodiment, the structure is thus no longer planar butflared, with an angle α defined between the top and bottom faces of theabove-mentioned plate. Thus, the angle of a guided mode thereforeincreases by 2a on each rebound of the guided mode from the bottom face.Applying the laws of geometrical optics to the successive images comingfrom a point on the top surface readily shows that the smallest angle(corresponding to the lowest effective index) increases up to the limitof 90° as the source generating guided waves approach the edge formed bythe intersection between the top and bottom faces of the waveguide.There therefore exists an ideal position between that edge and theoptofluidic system for placing the substantially non-directionalcoupling means.

In a particular version of this second embodiment, the top and bottomfaces of the waveguide are plane and the non-directional coupling meansare placed at one-fourth of the distance between an edge formed by theintersection of the top and bottom faces and the portion of thewaveguide carrying the chromophore elements.

In a possible variant, only the top face need be plane, it beingpossible for the bottom face to be curved and concave.

Preferably, the excitation light is coupled by diffusion and generatesguided modes that propagate in a plurality of directions inside thewaveguide.

The guided waves may be generated by illuminating a diffusing structureformed in or on the waveguide, thereby making it possible to form guidedwaves that propagate in a plurality of directions inside the waveguide,and avoiding a subsequent step of making the guided light uniform in theplane of the waveguide.

Advantageously, the diffusing structure used for providing substantiallynon-directional coupling is a structure having a disordered spatialdistribution of index.

The diffusing structure may be formed by frosting with a typical grainsize both in the plane of the waveguide and perpendicularly theretolying in the range 0.1 micrometers (μm) to 50 μm. The diffusingstructure may also be formed by a layer deposited on a face of thewaveguide, e.g., a layer of “Teflon” or of metallic or colloidalparticles.

In a variant, the diffusing structure may comprise diffusing particlesin a matrix of a resin, e.g., such as an acrylic resin, aglycerophthalic resin, or a polymer, which may be a fluoropolymer. Inorder to guarantee good diffusion of the excitation light by thediffusing structure, it is preferable for the matrix to have arefractive index that is less than that of the diffusing particles by atleast Dn=0.5. It is thus preferable to use particles of high index,e.g., oxides such as TiO2, Ta2O5, BaSO4.

The diffusing structure may also be situated inside the waveguide andmay be made in the form of microcavities having dimensions of the orderof 0.1 μm to 40 μm, and preferably of the order of 0.1 μm to 30 μm. Itmay also be made in the form of local modifications such as locallyforming non-stoichiometric compounds of the SiOx type in glass, forexample, or indeed in the form of molecular zones of phases differentfrom the phase of the waveguide, e.g., ordered instead of amorphous, inparticular. These stoichemetric changes or phase changes affect theindex or the dielectric tensor of the diffusing structure. Such adiffusing structure may be made by localized energy delivery by using alaser focused on the point at which it is desired to form the diffusingstructure.

In a second embodiment of the diffusing structure, the diffusingstructure is deposited on a face of the waveguide and comprises a layerof fluorophore material and responds to light excitation by generatingfluorescent light that propagates in turn in the waveguide in the formof waves having an evanescent portion.

The fluorophore materials may be of a very wide variety of kinds and inparticular they may comprise quantum dots, organic fluorophores, orfluorophores based on rare earth or on luminescent ions.

BRIEF DESCRIPTION OF THE DRAWINGS

Other advantages and characteristics of the invention appear on readingthe following description made by way of non-limiting example and withreference to the accompanying drawings, in which:

FIG. 1 is a diagrammatic section view of a prior biochip art device;

FIGS. 2 and 3 are diagrammatic section views of a biochip device of theinvention including mode filter means integrated in the waveguide;

FIG. 4 is a diagrammatic section view of a biochip device of theinvention in which the waveguide carries the mode filter means;

FIGS. 5 and 6 are diagrammatic section views of two variants of the FIG.4 device;

FIGS. 7A, 7A′, 7B, and 7C are diagrams of the portion of the waveguidewhere guided waves are formed having an evanescent portion; and

FIGS. 8 to 13 show various setups enabling guided waves to be generatedin the waveguide with the device of the invention.

DETAILED DESCRIPTION

Reference is made initially to FIG. 1 , which shows a prior art biochipdevice 10 comprising a substrate 12 including a top layer 14 forming awaveguide. An excitation light 16 is directed to coupling means 18,e.g., such as a grating formed on the surface of the waveguide so as tocause a guided wave to propagate inside the waveguide 14. At a distancefrom the grating 18, the waveguide 14 carries a hybridizing chamber 20containing a solution 22 including target molecules marked bychromophore elements and suitable for hybridizing with probe moleculesdeposited on pads 24 on the surface of the waveguide 14.

Detector means are provided, e.g., on the face of the substrate 12 thatis opposite from its face carrying the hybridizing chamber 20, and theycomprise a camera 26, such as a charge coupled device (CCD) or acomplementary metal oxide on silicon (CMOS) camera, and a filter 28 forrejecting the light for exciting the chromophore.

In such a device, the evanescent portion of the guided wave excites thechromophores carried by the waveguide 14. Nevertheless, and as mentionedabove, that type of device can be difficult to implement because of thedifficulty of achieving appropriate optical coupling between theincident light and the waveguide, since the coupling requires greatprecision on the collimation angle of incidence of the excitation lightif coupling is performed by a conventional resonant grating in guidedoptics, or else it requires submicron mechanical precision if thecoupling is directly via the edge face. Furthermore, and above all, whenthe waveguide is suitable for having a plurality of guided modespropagate therein, i.e., a waveguide typically having a thicknessgreater than the wavelength of the guided waves for index steps of about1, then guided waves having an effective index of less than the index ofthe elements surrounding the chromophores, such as the material of thehybridizing chamber 20 or the hybridizing solution 22, are extractedfrom the waveguide 14 and excite target molecules that are present inthe hybridizing solution 22 but that are not attached to probemolecules. This results in a decrease in the signal-to-noise ratio whenmeasurement of the luminescence emitted by the chromophores is performedin real time.

The device of the invention proposes using coupling means 19 that aresubstantially non-directional and of dimensions that are not verycritical (e.g., 0.1 millimeter (mm) to 10 mm) providing optical couplingbetween the light source and the waveguide so as to generate waves thatare guided in a plurality of directions inside the waveguide, e.g., froman excitation light beam that is not collimated. By way of example, suchcoupling means may be of diffusing structure and they are described ingreater detail below in the description.

Advantageously, the coupling means are combined with mode filter meansso as to extract from the multimode planar waveguide 14 those guidedmodes for which the effective index is less than or equal to apredetermined threshold value.

These mode filter means may either be formed by the waveguide itself(FIGS. 2 and 3 ) or else they may be carried thereby (FIGS. 4 to 6 ).

Reference is now made to FIG. 2 , which shows a stratified structurecomprising three superposed layers, with the top layer 30 serving as aplanar waveguide without absorption and having an index n1. The secondlayer 32 is interposed between the top layer 30 and an absorption bottomlayer 34. The extraction intermediate layer 32 has an index n2 that issubstantially equal to the predetermined threshold index value.

In this embodiment, the top layer 30 has the coupling means 19 at oneend and has an optofluidic portion 36 at another end. The intermediateand absorption layers 32 and 34 extend over the entire length of theguiding top layer 30 so as to perform filtering over the entire length Lbetween the substantially non-directional coupling means 19 and theoptofluidic portion 36.

The index n3 of the absorbent layer 34 is selected to be greater thann2, while the thickness of the layer is appropriate in application ofthe rules of guided optics and n1 is sufficiently large given thecontrast firstly with n2 and secondly with the index n of thehybridizing solution 22 to be capable of accepting at least one mode ofindex higher than the desired threshold. The thickness z2 of theintermediate layer 32 of index n2 is sufficient to ensure that this modedoes not escape over the distance L: the exponential decay factor in z2,f=exp[2×π×(z2/1)×{√(neff2−n2>1)} must be at least three times greaterthan the ratio L/z1 of L to the thickness z1 of the layer 30 of theindex n1 for the intermediate layer to perform its role over the lengthL. The modes of index neff<n2 are obliged to propagate in the absorbentthird layer of thickness z3 and to travel a distance of about L therein.It then suffices to provide this third layer 34 with an absorptioncoefficient α3 that is greater than 2/L in order to attenuate theundesirable modes of index neff<n2.

By way of example, these three stratified layers may be made of polymer.A typical index sequence may be n1=1.55, n2=1.42, and n3=1.55. Thethicknesses may be z1=5 μm to 50 μm for the layer 30, z2>3 μm for thelayer 32, and z3 greater than or equal to z1 for the layer 34, e.g.,being about 500 μm for a length L of 1 centimeter (cm). The absorptionmay be obtained by means of an organic or inorganic dye dispersed in thethird layer 34. In practice, in order to absorb the light at awavelength of 532 nanometers (nm) that is used for exciting chromophoressuch as Cyanine 3, it is possible to perform doping with absorbents thatare stable, such as Fe3+ salts of iron.

FIG. 3 shows a second embodiment of coupling means formed by thewaveguide. In this embodiment, the top and bottom faces 38 and 40 of thewaveguide 42 are plane and they diverge from each other going from thecoupling means 19 towards the optofluidic portion 36, e.g., in the formof a wedge. The top and bottom faces 38 and 40 of the waveguide 42 forman angle α at the edge 44 where they intersect, which edge in thepresent example is situated outside the device. Such a waveguide 42 isreferred to as a “wedge” waveguide. The light rays coming from thecoupling means 19 that are reflected on the bottom face 40 becomeincreasingly inclined relative to the top face 38, by an angle 2α foreach pair of reflections. This filtering is “dynamic” filtering in whichthe energy of the modes is shifted to ever increasing effective indicesas the light rays propagate.

In order to understand the operation of such filtering, it is possibleto make use of the laws of geometrical optics. It is thus possible topredict that the successive images 46 of the coupling means 19 areturned through successive angles of 2 a on each pair of reflections andthat they move away towards the edge 44 by following a semicirclecentered on the edge 44. This implies a limit on the angle of incidencethat can be achieved at the optofluidic portion, as a function of thetwo pertinent distances in the geometrical optics problem, namely L′corresponding to the distance of the edge 44 from the coupling means 19,and the distance L from the coupling means 19 to the optofluidic portion36. Constructing a right-angled triangle ABC with A at the edge 44 andof radius L′ shows that the maximum angle of incidence Θ of a light rayon the optofluidic portion is given by: sin(90°−Θ)=cos Θ=L′/(L+L′) sincethe right-angled triangle ABC has the angle 90°−Θ at the vertex C.Determining this angle thus requires a minimum value for the effectiveindex that is given by neff=n×sin(Θ). In practice, it is desirable toaim for angles Θ of about 70° (neff=1.41 for a waveguide material havingan index of about n=1.5). This imposes the following ratio:L/L′=1−1/sin(90°−arcsin(neff/n))=2. It should be observed that thisresult is independent of the angle α of the wedge. This angle may beselected in practice to lie in the range 2° to 10°. The overall extentof the coupling means 19, e.g., diffusers, needs to be taken intoaccount, seeking merely to ensure that the desired condition applies forthe most unfavorable of the light rays coming from said means, i.e.,those from the source 19 that are the closest to the hybridizing chamber20.

In other variants (not shown), the top and bottom faces may diverge fromeach other, but without being plane, as would be the case for examplewith curved faces that are concave. The top face may also be plane whilethe bottom face may be curved, for example it may present a concavecurve.

In the embodiments shown in FIGS. 4 to 6 , the filter means are carriedby the waveguide and they are interposed between the coupling means 19and the optofluidic portion 36 of the waveguide 14 carrying thehybridizing chamber 20. Advantageously, these means comprise anextraction layer 48 in contact with the top surface 50 that is a topface of the waveguide 52. The waveguide 52 has a bottom face 94 and aside wall 92 connecting the top surface 50 and the bottom face 94,illustrated in FIG. 5 . This extraction layer 48 is selected so that itsrefractive index is equal to the predetermined threshold value (FIG. 4).

Means 53 for absorbing or deflecting the guided modes that are extractedfrom the waveguide 52 are advantageously placed on the extraction layer48.

In a practical embodiment of the invention, absorption means may, forexample, comprise a filter 54 having a wide absorption spectrum thatperforms volume filtering on the guided wave. This type of filter isvery suitable since it reflects only very little of the guided lightwaves at its interface with the extraction layer 48 (FIG. 5 ).

Deflection means may, for example, comprise a prism 56 of index selectedto deflect the extracted guided light waves as shown at 58 in FIG. 6 .

Because of the presence of an extraction layer between the couplingmeans 19 and the optofluidic portion 36 of the waveguide 52 carrying thehybridizing chamber 20, the guided modes that are of effective indexthat is less than the index of the extraction layer 48 are extractedfrom the waveguide and are refracted inside said layer. The presence ofabsorption means 54 or deflection means 56 on the layer 48 prevents anyreflection of the guided modes that have been extracted via the topinterface of the extraction layer 48, which would lead to the extractedguided modes being re-introduced into the inside of the planar waveguide52.

In order to guarantee optimum filtering of the guided modes of effectiveindex less than the predetermined threshold value, the extraction layer48 and the absorption means 54 or the deflection means 56 must extend,between the zone 19 where the guided waves are generated and theoptofluidic portion 36 carrying the hybridizing chamber 20, over adistance that is greater than or equal to 2×e×tan Θ, where e is thethickness of the waveguide and Θ is the reflection angle inside thewaveguide relative to the normal to the waveguide 52 for the guided modefor filtering that has the greatest effective index. In this way, it ispossible to guarantee that all of the guided modes of effective indexless than the predetermined threshold value are subjected to at leastone reflection at the interface between the waveguide 52 and theextraction layer 48.

In a variant, the mode filter means may also extend at least in partunder the pads 24 in the hybridizing chamber so as to filter out thephotons that the edges of the chamber might diffuse towards uncontrolledindex modes of the waveguide that could then escape and excite thesolution.

In an embodiment of the invention, the hybridizing chamber 20 is made ofpolydimethylsiloxane (PDMS) having a refractive index n=1.42, and thehybridizing solution 22 is water-based having an index of about n=1.33.Under such circumstances, the extraction medium 48 is selected to havean index that is not less than the highest index in the environment ofthe chromophores, i.e., n=1.42.

In practice, the threshold index value is selected to lie in the rangen=1.30 to n=1.45, which corresponds to the index values commonlyencountered for the materials of the hybridizing chamber 20 and also forthe hybridizing solution 22.

There follows a more detailed description of the means used forgenerating a plurality of guided waves inside the planar waveguide andhaving an evanescent portion for exciting the chromophore elements fixedto the pads 24.

In a first embodiment, the device has a diffusing structure formed inthe waveguide or on the waveguide and that is to be illuminated by theexcitation light.

This diffusing structure (FIGS. 7 and 8 ) presents a disordered spatialdistribution of index so as to diffuse the excitation light into thewaveguide in a plurality of directions. Such a structure makes it easierto convert the excitation light into guided light and to do so withefficiency that depends little on the excitation conditions, and inparticular on the angle of incidence. Thus, with such a diffusingstructure, the angle of tolerance is quite large, being about 10degrees, which does not require a high precision mechanical and opticalsetup, as compared with tolerance of about 0.1 degrees when using agrating 18.

This diffusing structure may be on one or other of the faces of thewaveguide 52 that is transparent to the excitation light 16.

In a first embodiment of the diffusing structure 60, it is constitutedby a layer deposited on the waveguide 52 and presenting an internalstructure that is disordered. The layer may consist in a deposit ofmetallic or colloidal particles 60 (FIG. 7A), or indeed a deposit of“Teflon” (polytetrafluoroethylene) 61 (FIG. 7A′).

It is also possible to deposit a layer 62 made up of a matrix containingdiffusing particles 64 (FIG. 7B). The matrix may consist in a resin ofthe same type as that used for paints or varnishes, such as, forexample, acrylic resins or glycerophthalic resins or indeedfluoropolymers.

In order to guarantee good conversion by diffusion of the excitationlight 16 into guided waves with a thin diffusion layer, i.e., adiffusion layer with a thickness of about 15% to 60%, it is desirablefor the refractive index of the matrix to be less that the index of thediffusing particles 64 by at least Dn=0.5.

A diffusing structure 66 may also be obtained by making microcavities(FIG. 7C), e.g., spheroidal microcavities, inside the waveguide 52 or bylocally modifying the material of the waveguide 52 by changing itsdegree of oxidation or by changing its phase, from amorphous tocrystalline or from crystalline to amorphous, e.g., by means of laserpulses having a duration lying in the range 0.1 picoseconds (ps) to 1microsecond (ps) with typical energy lying in the range 1 nanojoule (nJ)to 100 to microjoules (pJ). This type of structure thus presents indexdiscontinuities suitable for diffusing the excitation light in aplurality of directions. There also exist methods of nucleating pores ina sol-gel phase, and these methods are often used for making layers oflow dielectric constant in microelectronics.

In another embodiment of a diffusing structure shown in FIG. 8 , itconsists in a layer 68 of fluorescent and diffusing material, such asthe phosphors of white light-emitting diodes (LEDs) that respond tolight excitation by generating guided waves having an evanescent portionand propagating in a plurality of directions.

The fluorescent material may consist in an ordered or disordered layerof fluorophores, in particular such as those based on quantum dots,organic fluorophores, or fluorophores based on rare earth.

The layer of fluorescent material may also consist in a layer comprisinga binder such as an organic or inorganic powder with grain size lying inthe range 0.1 μm to 50 μm or a polymer matrix belonging to the familiesused for makeup and for paint and varnishes, such as acrylic resins,glycerophthalic resins, etc., and fluorophore elements such as thosedescribed in the paragraph above.

The layer of fluorescent material may also include diffusing elements ofthe high-index particle type (e.g., oxides of titanium or carbonates ofcalcium or barium sulfate) and fluorophores such as those mentionedabove.

With the diffusing structure made in these ways, the process of creatingguided light waves possessing an evanescent portion may be thought of asemitting a set of optical dipoles at the surface of the waveguide orinside it. With a diffusing structure as shown in FIGS. 7A, 7A′, 7B, and7C, these dipoles oscillate at the same frequency as the excitationlight 16, and with a structure that diffuses by fluorescence, thedipoles oscillate after frequency conversion at the frequency at whichthe fluorophores fluoresce. Under such circumstances, the fluorescencefrequency is selected so as to be suitable for exciting the chromophorescarried by the pads 24.

The diffusing structure may be excited by the light 16 either directlyor indirectly, as shown in FIGS. 8 to 13 .

FIG. 8 shows excitation of the diffusing structure by transmission ofthe excitation light 16 through the diffusing structure 68. This ispossible only when the diffusing structure 68 presents little opaquenessin order to avoid the excitation light 16 being absorbed by thediffusing structure 68.

FIG. 9 shows the diffusing structure being excited by the excitationlight 16 being transited through the thickness of the waveguide 52.

In another configuration, shown in FIG. 10 , a prism 72 may be opticallycoupled to the waveguide 52 via an index-matching layer 74. It is alsopossible for the prism 72 to be integrated with the waveguide 52 bymolding. The excitation light 16 is thus deflected by the prism into theinside of the waveguide and it excites the diffusing structure 70.

In a similar embodiment, shown in FIG. 11 , a chamfer 76 is made at theend of the waveguide 52 that carries the diffusing structure 70. For thetwo embodiments shown in FIGS. 10 and 11 , the excitation light 16 isoriented parallel to the plane of the waveguide towards the slopingsurface of the prism 72 or of the chamfer 76. The light 16 is thendeflected towards the diffusing structure 70 in order to generate guidedlight waves having an evanescent portion.

In the embodiment of FIG. 12 , the diffusing structure 70 is carried bythe chamfered surface of the waveguide 52 and the excitation light maybe at any orientation relative to the plane of the waveguide 52 carryingthe pads 24. The excitation light 78 may thus be orientedperpendicularly to the waveguide 52 or it may be inclined relative tothe vertical to the waveguide 52 (as shown at 80). It passes through aportion of the waveguide in order to illuminate the diffusing structure70. The excitation light 82 may also excite the waveguide bydiffusion/transmission through the diffuser 70.

In a last embodiment, shown in FIG. 13 , the excitation light 16 istransmitted to the diffusing structure 70 through the polished orunpolished edge face of the waveguide 52.

1. A biochip device comprising: a stratified structure including a toplayer having a refractive index n₁, a bottom layer having a refractiveindex n₃, and an intermediate layer disposed between the top layer andthe bottom layer and having a refractive index n₂; a light coupleroptically coupling a light source and the top layer so as to generatewaves that are guided in a plurality of directions inside the top layer;and an optofluidic portion supported on a surface of the top layer andcomprising a hybridizing chamber containing a hybridizing solution andpads supported on the surface of the top layer and situated within thehybridizing chamber, wherein probe molecules are deposited on the pads,wherein the refractive index n₂ of the intermediate layer is greaterthan or equal to a highest index of refraction of the hybridizingchamber and the hybridizing solution.
 2. The biochip device of claim 1,wherein the refractive index n₃ of the bottom layer is greater than therefractive index n₂ of the intermediate layer.
 3. The biochip device ofclaim 1, further comprising chromophore elements deposited on the pads.4. The biochip device of claim 1, wherein the light coupler comprises alight diffusing structure.
 5. The biochip device of claim 4, wherein thelight diffusing structure comprises structure having a disorderedspatial distribution of refractive index.
 6. The biochip device of claim4, wherein the light diffusing structure comprises one of: frosting witha grain size of 0.1 μm to 50 μm; non-uniform metal or dielectricparticles, colloidal particles positioned in the top layer,micro-cavities formed within the top layer, and a layer ofpolytetrafluoroethylene on the top layer.
 7. The biochip device of claim4, wherein the light diffusing structure comprises diffusing particlesin a matrix of a resin.
 8. The biochip of claim 7, wherein the matrix isan acrylic resin, a glycerophthalic resin, or a fluoropolymer matrix. 9.The biochip device of claim 7, wherein the matrix has a refractive indexthat is less than the refractive index of the diffusing particles. 10.The biochip device of claim 7, wherein the diffusing particles compriseone of TiO₂, Ta₂O₅, and BaSO₄.
 11. The biochip device of claim 4,wherein the light diffusing structure comprises micro-cavities havingdimensions of 0.1 μm to 40 μm.
 12. The biochip device of claim 4,wherein the light diffusing structure is deposited on a face of the toplayer and comprises a layer of fluorophore material that responds tolight excitation by generating fluorescent light that propagates in thetop layer in the form of waves having an evanescent portion.
 13. Thebiochip device of claim 12, wherein the fluorophore material comprisesquantum dots, organic fluorophores, or fluorophores based on one of rareearth ions and luminescent ions.
 14. The biochip device of claim 1,wherein the light coupler is disposed at one end of the stratifiedstructure, and the optofluidic portion is disposed at an opposite end ofthe stratified structure.
 15. The biochip device of claim 1, wherein thelight coupler and the optofluidic portion are spaced apart, and whereinthe intermediate layer and the bottom layer extend over the entirelength of the top layer between the light coupler and the optofluidicportion.
 16. The biochip device of claim 1, wherein the light couplerand the optofluidic portion are spaced apart, and wherein the bottomlayer is formed of a material having an absorption coefficient α₃ thatis greater than 2/L, where “L” is a distance between the light couplerand the optofluidic portion.
 17. The biochip device of claim 16, whereinL is about 1 cm.
 18. The biochip device of claim 1, wherein thestratified structure is made of a polymer.
 19. The biochip device ofclaim 1, wherein n₁=1.55, n₂=1.42, and n₃=1.55.
 20. The biochip deviceof claim 1, wherein the top layer has a thickness z₁ is equal to 5 μm to50 μm, the intermediate layer has a thickness z₂ that is greater than 3μm, and the bottom layer has a thickness z₃ that is greater than orequal to z₁.
 21. The biochip device of claim 20, wherein z₃ is about 500μm.
 22. The biochip device of claim 1, wherein the bottom layercomprises an organic or inorganic dye dispersed in the bottom layer.